The present invention relates to newly designed plastic and disposable cell culture devices for widespread use in especially small-scale research laboratories and more particularly relates to functionally-oriented disposable devices integrated with micro-constructed plastic injected cell-culture embodiments generated by a variety of newly-designed micro-architectural engineering. The present invention finds particular utility in facilitating widespread, massive in vitro growth of micro-organisms, plant and animal cells, including germ cells, short and long-term tissue cultures, hybridomas and other biologically engineered cells, to maximize the in vitro yield of cell constituents and products such as monoclonal antibodies or other biologic agents, improve the exposure of cells to various agents, facilitate the investigation of cell to cell interaction improving cell transportation, long-term cell preservation, cell freezing and thawing.
Cell culture refers to the process by which living cells from a variety of sources, animal or plant, are grown and maintained alive in laboratory devices, using artificial culture media to support their basic biological functions. This procedure has become one of the cornerstones in modern animal and plant biology, basic and applied medical research, medical diagnosis and therapy, microbiology, genetic engineering and biotechnology.
Cell culture is also the keystone of in vitro cell fusion technologies, whereby parental cells are fused, either by a chemical or electrical process, or through other fusing processes, to yield newly-formed cells with integrated cytoplasms and nuclei with new biological capabilities, secretory or others, for a variety of purposes. The hybridoma technology is an excellent example whereby parental myeloma cells are fused with parental lymphocytes, to yield immortalized cells called hybridomas, capable of secreting monoclonal antibodies or other cellular factors in endless amounts and high purity. Cell culture is fundamental in other biological fields such as in cellular transfection and genetic engineering, as genes from cells, viruses or other sources are obtained by gene-splitting technologies and then transferred into new hosts to induce massive production of the encoded molecules.
The current art technologies involved for the in vitro growth of cells allow short and long-term maintenance of cells in tissue culture medium, expose cells to a variety of agents, study cell to cell interaction long-term cell preservation by freezing, to yield upon thawing high percentages of viable and functioning cells. Cell cloning is another common cell culture procedure by which heterogenous cell populations are separated by appropriate dilutions into homogenous clones each originating from one parental cell through multiple divisions. Screening efforts of the numerous homogenous clones enables the identification of minute amounts of cell constituents and products present within the waste cell-culture medium, specific for certain clones and thereafter their isolation, purification, characterization and utilization.
The spectrum of modern cell culture procedures can be illustrated by the technology of generating hybridoma clones secreting valuable monoclonal antibodies. The following refers to mouse hybridomas as well as human, rat and other species of hybridomas. While the initial steps involve optimal growth of the parental cell lines and their fusion in the presence of selective medium, the next crucial phases are the screening of cell containing newly-fused clones for their capacity to secrete useful antibodies. Once clones have been selected, the next steps are repeated cloning in the presence or absence of feeder layer cells, expanding the chosen clones, and ultimately obtaining a supernate containing high concentrations of the antibody.
Since hybridoma cell-culture waste medium such as obtained by mouse hybridomas contains limited amounts of antibody, researchers choose to inject hybridoma cells into the abdominal cavity of lipid-primed animals, as illustrated by mice, to induce abdominal fluid called ascitis, highly enriched with monoclonal antibody. While accumulating these hybridoma cells in their abdomens, and while being tapped almost daily to obtain the antibody containing ascitic fluid, these animals gradually become extremely heavy and within a week to three weeks or so stop moving in their cages, cannot reach their water bottles or food, and ultimately die in agony. This is repeated a number of times with numerous mice for each hybridoma clone, and the total number of mice depends on the number of different clones available in the specific laboratory. This procedure is widely used in small-scale research laboratories as well as in large-scale industrial laboratories, and it is estimated that more than thousands of mice and other animals per year are thus utilized for this purpose throughout the scientific community.
Because of a number of causes including rejection phenomena, many types of hybridomas such as human, rabbit, rat and so forth, will not grow within mice or other animals and therefore high titer, monoclonal antibody containing, ascitic fluid cannot be formed in vivo. In these instances the researcher is dependent on tissue culture scale-up procedures depending on the present state of the art. The present invention overcomes these problems and disadvantages of the prior art by generating novel microembodiment containing cell culture devices, enabling every researcher in every small laboratory to generate extremely high titers of antibodies or other cell products, circumventing the necessity of employing abdominal cavities of animals.
Furthermore, this in vitro new art has further advantages as ascitic fluid derived monoclonal antibody is contaminated by a huge variety of animal-derived proteins and other molecules, whereas the scale up procedures involving the present invention are free of this disadvantage.
Once a valuable hybridoma clone has been generated, its long term preservation is of great importance scientifically and economically. Early cell batches of the hybridoma clones are frozen at extremely low temperatures (up to--180.degree. C. or more) depending on the equipment available for freezing. Freezing procedures are harmful and can cause injury to a varying proportion of the cells. It is the objective of the biologist to preserve the viability of as many cells as possible whether using agents such as DMSO (Dimethyl sulfoxide), time-controlled gradual drop in temperature and other means. Current art allows cells to be frozen in monoplane devices such as tubes with flat or round bottom surfaces, and the present invention contributes significant advantages compared to the old art.
Occasionally cell transportation is required between laboratories, and attention is given to preserve their adequacy, sterility and stability. Cells can be transported in a frozen state using dry ice or in other freezing facilities. However, adequate low temperatures cannot be kept for long periods of time such as required when transportation is between countries or continents. Another alternative is transporting viable cells placed within small monoplane tubes or flasks treated with culture medium and kept in room temperature. The above cell culture procedures, as illustrated by the processing hybridoma clones, are essential for other types of cells used in modern biology, or biotechnological experimentation. All above cell maintenance processes are carried out in small-scale pilot research laboratories using disposable uniplanar labware. The present invention comprising a family of newly designed cell handling devices again contributes novel advantages for cell transportation, whether between continents in conventional transportation facilities, or, in the future in space vehicles, into outer space.
The growing of hybridoma cells in abdominal cavities of mice to generate ascitic fluid enriched with monoclonal antibodies is an exception and is associated with the agony caused to the animals used as hosts. Large-scale cell culture facilities employed in biotechnological plants are, by their nature, different from those used in the ordinary small-scale pilot research laboratory. The main goal of the former is massive production of cells and their products for industrial purposes and involves complicated and extremely expensive equipment, demands numerous personnel and is useless for the typical small laboratories engaged in early research and characterization. The unique and outstanding feature of the present invention is the small size simplicity and handiness of the proposed new devices.
Furthermore, the present invention is providing new tools to scientists and will facilitate cellular biology research, particularly cell to cell interation, cell migration, chemotaxis and separation with the identification of novel cell subsets, improved cell exposure to biological, chemical and other agents, cell transformations, transfection and mutation formation, cell fusion technology, in vitro fertilization, and a variety of other cellular behavioral phenomena.
The large-scale facilities utilize multiple roller bottles which are expensive, require intensive labor and carry a high risk of contamination. Other large scale facilities are aimed at increasing the surface of the containers used to grow cells such as roller bottles with one or more annular members placed inside the bottle which are described in U.S. Pat. No. 4,327,886, or a flexible strip wound into a compact cell support to fit inside the roller bottle as described in U.S. Pat. No. 3,853,712, or the use of a plurality of tubes clamped together and fixed to a shaft the entire device being rotated about a shaft placed within a roller-bottle-like apparatus as described in U.S. Pat. No. 3,732,149. Other devices for mass production are disclosed in U.S. Pat. No. 3,827,943 which employs individual tubes to increase the surface, or U.S. Pat. No. 4,514,499 which employs a monolithic support within an assembled composite to immobilize cells with flow providing means. Small scale disposable devices for cell cultures are described in U.S. Pat. No. 4,495,289 and U.S. Pat. No. 3,649,464, both describing multi-well plates all having uniplanar bottoms to grow cells.
One way of increasing the surface area in conventional roller bottles or cell culture devices is to utilize microcarrier beads, artificial capillaries (hollow fibers) and bundle tubes. Microcarrier culture system involve the suspension of millions of individual, minute beads made of gelatin or other materials within tissue culture devices and are not related directly to the present invention. It is worthwhile to notice that these beads are movable, interfere with the direct inspection of the flasks both macroscopically and microscopically and render an increased risk of contamination.